P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that plays a critical role in drug distribution, drug-drug interactions and Drug resistance. Drugs that modulate P-gp activity could have therapeutic utility by increasing delivery of other drugs to the central nervous system, or by improving their pharmacokinetic properties. P-gp is an extremely promiscuous transporter that includes a drug binding site, or sites, within a 12- transmembrane helix domain (TMs), which communicates with nucleotide binding domains (NBDs) that bind and hydrolyze ATP to drive conformational changes in the TM domain. Due to the difficulty in studying P-gp in model membranes no kinetic measurements have been made on any elementary step within the P-gp reaction cycle, which remains poorly defined. Knowledge of the rates of these processes is essential to determine which mechanistic models are most accurate, among several proposed schemes. The goals of this proposal are to exploit P-gp in lipid bilayer nanodiscs, to challenge two competing mechanistic models of P-gp with studies that utilize stopped-flow spectroscopy, surface plasmon resonance and single molecule total internal fluorescence microscopy (TIRFM). For the latter two methods, several surface attachment strategies will be compared to optimize data acquisition. The binding and dissociation rate constants of nucleotides will be determined in the presence of several P-gp substrates and inhibitors. An extension of these studies includes the use of single cysteine P-gp mutants covalently adducted with varying drugs, to determine the effect of drug location on nucleotide binding and dissociation. Similarly, binding and dissociation of fluorescent drugs will be determined with varying nucleotides bound at the NBDs. These studies will provide the first kinetic data for P-gp ligand interactions that will elucidate several mechanistic details that have not been previously clarified. Such studies could lead to conformation-specific inhibitors of P-gp with utility in the modulation of pharmacokinetic properties of existing drugs. PUBLIC HEALTH RELEVANCE: P-glycoprotein is a large transmembrane helical efflux transporter that plays a major role in cancer cell resistance to drugs and the distribution and clearance of most drugs spanning all therapeutic areas. The molecular mechanism of P-gp remains unknown. The long term goal of this proposal is to understand the molecular mechanisms of the efflux transporter P-glycoprotein in order to rationally design drugs that target P-gp and improve the therapeutic profile of other drugs.